www.nature.com/scientificreports
OPEN
received: 25 February 2016 accepted: 11 May 2016 Published: 01 June 2016
Hyperlipidemia-associated gene variations and expression patterns revealed by whole-genome and transcriptome sequencing of rabbit models Zhen Wang1,*, Jifeng Zhang2,*, Hong Li1,*, Junyi Li1,*, Manabu Niimi3, Guohui Ding1,4, Haifeng Chen4,5, Jie Xu2, Hongjiu Zhang6, Ze Xu7, Yulin Dai8,9, Tuantuan Gui8,9, Shengdi Li8,9, Zhi Liu8,9, Sujuan Wu4,10, Mushui Cao4,11, Lu Zhou8,9, Xingyu Lu5, Junxia Wang5, Jing Yang4,10, Yunhe Fu8,9, Dongshan Yang2, Jun Song2, Tianqing Zhu2, Shen Li3, Bo Ning3, Ziyun Wang3, Tomonari Koike12, Masashi Shiomi12, Enqi Liu13,14, Luonan Chen8, Jianglin Fan3,14, Y. Eugene Chen2 & Yixue Li1,4,5,10,11 The rabbit (Oryctolagus cuniculus) is an important experimental animal for studying human diseases, such as hypercholesterolemia and atherosclerosis. Despite this, genetic information and RNA expression profiling of laboratory rabbits are lacking. Here, we characterized the whole-genome variants of three breeds of the most popular experimental rabbits, New Zealand White (NZW), Japanese White (JW) and Watanabe heritable hyperlipidemic (WHHL) rabbits. Although the genetic diversity of WHHL rabbits was relatively low, they accumulated a large proportion of high-frequency deleterious mutations due to the small population size. Some of the deleterious mutations were associated with the pathophysiology of WHHL rabbits in addition to the LDLR deficiency. Furthermore, we conducted transcriptome sequencing of different organs of both WHHL and cholesterol-rich diet (Chol)-fed NZW rabbits. We found that gene expression profiles of the two rabbit models were essentially similar in the aorta, even though they exhibited different types of hypercholesterolemia. In contrast, Chol-fed rabbits, but not WHHL rabbits, exhibited pronounced inflammatory responses and abnormal lipid metabolism in the liver. These results provide valuable insights into identifying therapeutic targets of hypercholesterolemia and atherosclerosis with rabbit models.
1
Key Lab of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. 2Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, MI, USA. 3Department of Molecular Pathology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi, Japan. 4Shanghai Center for Bioinformation Technology, Shanghai Industrial Technology Institute, Shanghai, China. 5School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai, China. 6Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA. 7EG Information Technology Enterprise (EGI), BasePair Biotechnology Co., Ltd., Shanghai, China. 8Key Lab of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. 9University of Chinese Academy of Sciences, Beijing, China. 10School of Biotechnology, East China University of Science and Technology, Shanghai, China. 11School of Life Science and Technology, Shanghai Tongji University, Shanghai, China. 12Institute for Experimental Animals, Kobe University School of Medicine, Kobe, Japan. 13 Research Institute of Atherosclerotic Disease and Laboratory Animal Center, Xi’an Jiaotong University School of Medicine, Xi’an, China. 14Department of Pathology, Xi’an Medical University, Xi’an, China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to J.F. (email: jianglin@ yamanashi.ac.jp) or Y.E.C. (email: [email protected]) or Y.L. (email: [email protected]) Scientific Reports | 6:26942 | DOI: 10.1038/srep26942
1
www.nature.com/scientificreports/ DNA Breed
NZW
Sample size
Depth of coverage per sample
n = 10
13.34
Nucleotide diversity π
2.80 × 10−3
RNA Watterson θ
2.26 × 10−3
Tajima D
Tissue collected
Sample size
Data (Gb) per tissue per sample
Standard chow diet
Aorta, liver, heart, kidney
n = 4
5.10
0.3% cholesterol diet
Aorta, liver, heart, kidney
n = 4
5.13
Diet
0.77
JW
n = 10
12.94
1.61 × 10−3
1.37 × 10−3
−0.01
Standard chow diet
Aorta, liver, heart, kidney
n = 4
5.93
WHHL
n = 10
12.75
1.44 × 10−3
1.19 × 10−3
−0.08
Standard chow diet
Aorta, liver, heart, kidney
n = 4
5.70
Table 1. Experimental design and sequencing data.
The European rabbit (Oryctolagus cuniculus) is an important experimental animal model for biomedical science. Rabbits are not only the most-used animal for the production of antibodies, but also they are widely used for studying a variety of human diseases, such as infectious disease, neoplasms, ophthalmic disease, Alzheimer’s disease, and respiratory disease1. Like humans, but unlike rodents, such as mice and rats, rabbits have unique features of lipid metabolism that have made them an important model for human hyperlipidemia and atherosclerosis2. The first experiment using rabbits to investigate atherosclerosis was performed more than a century ago3. When fed a diet rich in cholesterol, laboratory rabbits rapidly develop hypercholesterolemia and atherosclerosis2. In addition, genetic defects in low density lipoprotein receptor (LDLR) in Watanabe heritable hyperlipidemic (WHHL) rabbits can lead to spontaneous hypercholesterolemia and atherosclerosis, even when they are on a normal chow diet4,5. Therefore, the rabbit model has provided tremendous breakthroughs and insights into understanding the molecular and cellular mechanisms of atherosclerosis, including the discoveries of LDLR deficiency as a cause for human familial hypercholesterolemia6 and statin, the most potent lipid-lowering drug7, which is prescribed annually for more than 30 million hyperlipidemic patients worldwide8,9. Despite the importance of rabbit models for the study of hypercholesterolemia and atherosclerosis, genomic and transcriptomic information related to hyperlipidemia and atherosclerosis is lacking, which hampers the use of rabbits for translational research2. Recently, a high-quality reference genome for the European rabbit with references to domestication and speciation was reported10,11. In the current study, we performed whole-genome sequencing on three breeds of popular experimental rabbits, wild-type New Zealand White (NZW), Japanese White (JW) and WHHL rabbits, in an attempt to identify whether there are other gene mutations or modifiers that may be involved in the pathogenesis of hypercholesterolemia and atherosclerosis in WHHL rabbits. In addition to WHHL rabbits, cholesterol-rich diet (Chol)-fed rabbits are often used as a model for the study of human hypercholesterolemia and atherosclerosis2. While both WHHL and Chol-fed rabbits exhibit hypercholesterolemia and atherosclerosis, the gene expression profiles of atherosclerotic lesions and livers have not been systemically investigated. Toward this goal, we conducted deep transcriptome sequencing of the aortas, livers, hearts and kidneys derived from the two hypercholesterolemic models along with wild-type control rabbits. These results provide valuable resources for the investigation of hypercholesterolemia and atherosclerosis using rabbit models.
Results
Whole-genome sequencing of laboratory rabbits. We collected three common breeds of laboratory
rabbits: NZW, JW and WHHL rabbits (Table 1). On a standard chow diet, both NZW and JW rabbits have relatively low plasma cholesterol levels compared to humans, and their cholesterol is mainly carried by high density lipoproteins (HDLs, Fig. 1a). WHHL rabbits are genetically deficient in LDLR function; thus, they develop hypercholesterolemia and atherosclerosis, even on a standard chow diet. Normal rabbits can also develop hypercholesterolemia and atherosclerosis when fed a diet rich in cholesterol (Chol). Although both WHHL and Chol-fed rabbits exhibit hypercholesterolemia, their lipoprotein profiles are quite different; WHHL rabbits have increased levels of LDL-cholesterol accompanied by low HDLs, while Chol-fed rabbits have increased hepatically and intestinally derived remnant lipoproteins, called β-VLDL (Fig. 1a). We performed whole-genome sequencing of 10 rabbits for each of the three breeds (Supplementary Fig. S1), resulting in a depth of coverage of approximately 13×for each individual after alignment to the reference genome (Fig. 1b and Supplementary Fig. S2). Totally, we identified 29.8 million SNPs (Supplementary Fig. S3) and 1.6 million small indels (Supplementary Fig. S4) in the 30 genomes. Phylogenic tree building (Fig. 1c) and principal component analysis (Supplementary Fig. S5) based on genome-wide SNPs conformed distinct genetic backgrounds of the three breeds. Most of the rabbits were assumed to be unrelated except two pairs of WHHL rabbits (Supplementary Fig. S5). The genetic diversity of NZW rabbits (Table 1), measured by the nucleotide diversity π (Fig. 1d) and Watterson’s θ (Supplementary Fig. S6), was consistent with a recent report11. It was also higher than that of JW and WHHL rabbits, suggesting that even though all of the breeds originated from European rabbits, NZW rabbits are derived from a larger population of progenitors. Furthermore, the Tajima’s D (Table 1 and Supplementary Fig. S6) of NZW rabbits was positive and the largest among the three breeds, suggesting a moderate population bottleneck (sharp reduction in population size) during domestication12. In contrast, the Tajima’s D of WHHL rabbits was negative and the smallest value, which is consistent with the fact that the breed Scientific Reports | 6:26942 | DOI: 10.1038/srep26942
2
www.nature.com/scientificreports/
Figure 1. Whole-genome sequencing of laboratory rabbits. (a) Lipid profiles of standard chow-fed NZW, Chol-fed NZW and WHHL rabbits analyzed by high performance liquid chromatography. The Chol-fed NZW showed elevated β-VLDLs, and the WHHL rabbits showed increased LDLs and reduced HDLs. (b) Cumulative distribution of depth of coverage for whole-genome sequencing. The average depth of coverage was 13× for each individual rabbit. (c) Phylogenic tree of the rabbits. The tree was constructed on the basis of representative SNPs with the maximum likelihood method. Bootstrap values are marked on the branch. (d) Distribution of nucleotide diversity π. The statistics were calculated for every 100 kb sliding-window across the genome.
underwent a severe population bottleneck during artificial selection. The level of linkage disequilibrium was lowest in NZW rabbits and highest in WHHL rabbits (Supplementary Fig. S7), which is also in agreement with their breeding history.
Deleterious mutations in WHHL rabbits. Although WHHL rabbits are well-known for their LDLR
mutation as a cause of hypercholesterolemia5, it is possible that other deleterious mutations could rise to high frequency by genetic drift due to the extremely small population size of the breed. To search for such deleterious mutations possibly involved in cardiovascular diseases, we compiled a comprehensive gene list associated with cardiovascular diseases from both the knowledge database and human genome-wide association studies (Supplementary Fig. S8). Based on the functional annotations of SNPs and indels, we predicted deleterious mutations in the prior genes using the following criteria: 1) the mutation should alter the protein sequence; 2) for non-synonymous SNPs, it should have a SIFT score 0.4 WHHL rabbits harbor a larger proportion of deleterious mutations with high frequency than NZW rabbits, which supported our hypothesis that deleterious mutations were easier to accumulate in the WHHL breed. As the genetic diversity and hence the population size of JW rabbits were quite close to WHHL rabbits (Table 1), this result could only be observed when ΔAF > 0.8 between the two breeds. We focused on the putative deleterious mutations specifically enriched in WHHL rabbits with a criterion of ΔAF > 0.7 compared with both normal breeds, which resulted in 24 deleterious mutations in the prior genes in addition to the known 12-bp in-frame deletion in LDLR5 (Fig. 2b and Supplementary Table S1). One of the deleterious mutations was located in ALDH2, the activation of which was found to decrease aortic atherosclerosis14. It is well-known that a loss-of-function mutation in human ALDH2, E487K, causes alcohol flushing and is associated with an increased risk of cardiovascular diseases15. Similar to this mutation in humans, the deleterious mutation that we predicted in the WHHL rabbits, R99C, occurred at a conserved site, which was invariant among all the vertebrates we examined (Fig. 2c and Supplementary Fig. S9). The mutant allele frequency remained low in the normal rabbits (NZW 10%, JW 0%) but became fixed in WHHL rabbits (100%). Another putative deleterious mutation was located in VWF (Fig. 2b), the mediator of blood coagulation. A mutation at the same position in the human VWF protein was reported in patients with type I von Willebrand’s disease16. As a hypercoagulable state was found in WHHL rabbits17, the role of the deleterious mutation of VWF deserves further investigation. There Scientific Reports | 6:26942 | DOI: 10.1038/srep26942
3
www.nature.com/scientificreports/
Figure 2. Deleterious mutations in WHHL rabbits. (a) Proportion of deleterious mutation by difference in allele frequency (ΔAF) between WHHL and NZW or JW rabbits. (b) Putative deleterious mutations in WHHL rabbits with ΔAF > 0.7 compared with both normal rabbits. Colors show the density of SNPs from high (red) to low (blue). Genes harboring deleterious mutations are highlighted. (c) Non-synonymous mutations in ALDH2. The red cross indicates the locations of mutations. R99C is a putative deleterious mutation in WHHL rabbits. E487K is a known loss-of-function mutation in humans. Both mutations occur at highly conserved sites across vertebrates.
was also a deleterious mutation identified in OLR1 (Fig. 2b), and the gene was highly expressed in the atherosclerotic lesions of WHHL rabbits18. These results suggested that the deleterious mutations could function as genetic modifiers in the pathophysiology of WHHL rabbits.
Transcriptome profiling of WHHL and Chol-fed rabbits. We conducted deep transcriptome sequencing of different organs collected from both normal and hypercholesterolemic rabbits (Table 1 and Supplementary Figs S10 and 11). More than 77% of the assembled transcripts were isoforms of 15,760 known genes recorded in Ensembl (v76)19 (Supplementary Fig. S12). Samples originating from the same tissue were clustered together based on their gene expression profiles (Supplementary Fig. S13). We performed differential expression analysis for Chol-fed versus normal chow-fed NZW rabbits and WHHL versus JW rabbits (Supplementary Figs S14 and 15). The results showed that differentially expressed genes (DEGs) predominantly occurred in the aortas of both hypercholesterolemic models (2,719 and 1,627, respectively, false discovery rate [FDR]
OPEN
received: 25 February 2016 accepted: 11 May 2016 Published: 01 June 2016
Hyperlipidemia-associated gene variations and expression patterns revealed by whole-genome and transcriptome sequencing of rabbit models Zhen Wang1,*, Jifeng Zhang2,*, Hong Li1,*, Junyi Li1,*, Manabu Niimi3, Guohui Ding1,4, Haifeng Chen4,5, Jie Xu2, Hongjiu Zhang6, Ze Xu7, Yulin Dai8,9, Tuantuan Gui8,9, Shengdi Li8,9, Zhi Liu8,9, Sujuan Wu4,10, Mushui Cao4,11, Lu Zhou8,9, Xingyu Lu5, Junxia Wang5, Jing Yang4,10, Yunhe Fu8,9, Dongshan Yang2, Jun Song2, Tianqing Zhu2, Shen Li3, Bo Ning3, Ziyun Wang3, Tomonari Koike12, Masashi Shiomi12, Enqi Liu13,14, Luonan Chen8, Jianglin Fan3,14, Y. Eugene Chen2 & Yixue Li1,4,5,10,11 The rabbit (Oryctolagus cuniculus) is an important experimental animal for studying human diseases, such as hypercholesterolemia and atherosclerosis. Despite this, genetic information and RNA expression profiling of laboratory rabbits are lacking. Here, we characterized the whole-genome variants of three breeds of the most popular experimental rabbits, New Zealand White (NZW), Japanese White (JW) and Watanabe heritable hyperlipidemic (WHHL) rabbits. Although the genetic diversity of WHHL rabbits was relatively low, they accumulated a large proportion of high-frequency deleterious mutations due to the small population size. Some of the deleterious mutations were associated with the pathophysiology of WHHL rabbits in addition to the LDLR deficiency. Furthermore, we conducted transcriptome sequencing of different organs of both WHHL and cholesterol-rich diet (Chol)-fed NZW rabbits. We found that gene expression profiles of the two rabbit models were essentially similar in the aorta, even though they exhibited different types of hypercholesterolemia. In contrast, Chol-fed rabbits, but not WHHL rabbits, exhibited pronounced inflammatory responses and abnormal lipid metabolism in the liver. These results provide valuable insights into identifying therapeutic targets of hypercholesterolemia and atherosclerosis with rabbit models.
1
Key Lab of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. 2Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, MI, USA. 3Department of Molecular Pathology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi, Japan. 4Shanghai Center for Bioinformation Technology, Shanghai Industrial Technology Institute, Shanghai, China. 5School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai, China. 6Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA. 7EG Information Technology Enterprise (EGI), BasePair Biotechnology Co., Ltd., Shanghai, China. 8Key Lab of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. 9University of Chinese Academy of Sciences, Beijing, China. 10School of Biotechnology, East China University of Science and Technology, Shanghai, China. 11School of Life Science and Technology, Shanghai Tongji University, Shanghai, China. 12Institute for Experimental Animals, Kobe University School of Medicine, Kobe, Japan. 13 Research Institute of Atherosclerotic Disease and Laboratory Animal Center, Xi’an Jiaotong University School of Medicine, Xi’an, China. 14Department of Pathology, Xi’an Medical University, Xi’an, China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to J.F. (email: jianglin@ yamanashi.ac.jp) or Y.E.C. (email: [email protected]) or Y.L. (email: [email protected]) Scientific Reports | 6:26942 | DOI: 10.1038/srep26942
1
www.nature.com/scientificreports/ DNA Breed
NZW
Sample size
Depth of coverage per sample
n = 10
13.34
Nucleotide diversity π
2.80 × 10−3
RNA Watterson θ
2.26 × 10−3
Tajima D
Tissue collected
Sample size
Data (Gb) per tissue per sample
Standard chow diet
Aorta, liver, heart, kidney
n = 4
5.10
0.3% cholesterol diet
Aorta, liver, heart, kidney
n = 4
5.13
Diet
0.77
JW
n = 10
12.94
1.61 × 10−3
1.37 × 10−3
−0.01
Standard chow diet
Aorta, liver, heart, kidney
n = 4
5.93
WHHL
n = 10
12.75
1.44 × 10−3
1.19 × 10−3
−0.08
Standard chow diet
Aorta, liver, heart, kidney
n = 4
5.70
Table 1. Experimental design and sequencing data.
The European rabbit (Oryctolagus cuniculus) is an important experimental animal model for biomedical science. Rabbits are not only the most-used animal for the production of antibodies, but also they are widely used for studying a variety of human diseases, such as infectious disease, neoplasms, ophthalmic disease, Alzheimer’s disease, and respiratory disease1. Like humans, but unlike rodents, such as mice and rats, rabbits have unique features of lipid metabolism that have made them an important model for human hyperlipidemia and atherosclerosis2. The first experiment using rabbits to investigate atherosclerosis was performed more than a century ago3. When fed a diet rich in cholesterol, laboratory rabbits rapidly develop hypercholesterolemia and atherosclerosis2. In addition, genetic defects in low density lipoprotein receptor (LDLR) in Watanabe heritable hyperlipidemic (WHHL) rabbits can lead to spontaneous hypercholesterolemia and atherosclerosis, even when they are on a normal chow diet4,5. Therefore, the rabbit model has provided tremendous breakthroughs and insights into understanding the molecular and cellular mechanisms of atherosclerosis, including the discoveries of LDLR deficiency as a cause for human familial hypercholesterolemia6 and statin, the most potent lipid-lowering drug7, which is prescribed annually for more than 30 million hyperlipidemic patients worldwide8,9. Despite the importance of rabbit models for the study of hypercholesterolemia and atherosclerosis, genomic and transcriptomic information related to hyperlipidemia and atherosclerosis is lacking, which hampers the use of rabbits for translational research2. Recently, a high-quality reference genome for the European rabbit with references to domestication and speciation was reported10,11. In the current study, we performed whole-genome sequencing on three breeds of popular experimental rabbits, wild-type New Zealand White (NZW), Japanese White (JW) and WHHL rabbits, in an attempt to identify whether there are other gene mutations or modifiers that may be involved in the pathogenesis of hypercholesterolemia and atherosclerosis in WHHL rabbits. In addition to WHHL rabbits, cholesterol-rich diet (Chol)-fed rabbits are often used as a model for the study of human hypercholesterolemia and atherosclerosis2. While both WHHL and Chol-fed rabbits exhibit hypercholesterolemia and atherosclerosis, the gene expression profiles of atherosclerotic lesions and livers have not been systemically investigated. Toward this goal, we conducted deep transcriptome sequencing of the aortas, livers, hearts and kidneys derived from the two hypercholesterolemic models along with wild-type control rabbits. These results provide valuable resources for the investigation of hypercholesterolemia and atherosclerosis using rabbit models.
Results
Whole-genome sequencing of laboratory rabbits. We collected three common breeds of laboratory
rabbits: NZW, JW and WHHL rabbits (Table 1). On a standard chow diet, both NZW and JW rabbits have relatively low plasma cholesterol levels compared to humans, and their cholesterol is mainly carried by high density lipoproteins (HDLs, Fig. 1a). WHHL rabbits are genetically deficient in LDLR function; thus, they develop hypercholesterolemia and atherosclerosis, even on a standard chow diet. Normal rabbits can also develop hypercholesterolemia and atherosclerosis when fed a diet rich in cholesterol (Chol). Although both WHHL and Chol-fed rabbits exhibit hypercholesterolemia, their lipoprotein profiles are quite different; WHHL rabbits have increased levels of LDL-cholesterol accompanied by low HDLs, while Chol-fed rabbits have increased hepatically and intestinally derived remnant lipoproteins, called β-VLDL (Fig. 1a). We performed whole-genome sequencing of 10 rabbits for each of the three breeds (Supplementary Fig. S1), resulting in a depth of coverage of approximately 13×for each individual after alignment to the reference genome (Fig. 1b and Supplementary Fig. S2). Totally, we identified 29.8 million SNPs (Supplementary Fig. S3) and 1.6 million small indels (Supplementary Fig. S4) in the 30 genomes. Phylogenic tree building (Fig. 1c) and principal component analysis (Supplementary Fig. S5) based on genome-wide SNPs conformed distinct genetic backgrounds of the three breeds. Most of the rabbits were assumed to be unrelated except two pairs of WHHL rabbits (Supplementary Fig. S5). The genetic diversity of NZW rabbits (Table 1), measured by the nucleotide diversity π (Fig. 1d) and Watterson’s θ (Supplementary Fig. S6), was consistent with a recent report11. It was also higher than that of JW and WHHL rabbits, suggesting that even though all of the breeds originated from European rabbits, NZW rabbits are derived from a larger population of progenitors. Furthermore, the Tajima’s D (Table 1 and Supplementary Fig. S6) of NZW rabbits was positive and the largest among the three breeds, suggesting a moderate population bottleneck (sharp reduction in population size) during domestication12. In contrast, the Tajima’s D of WHHL rabbits was negative and the smallest value, which is consistent with the fact that the breed Scientific Reports | 6:26942 | DOI: 10.1038/srep26942
2
www.nature.com/scientificreports/
Figure 1. Whole-genome sequencing of laboratory rabbits. (a) Lipid profiles of standard chow-fed NZW, Chol-fed NZW and WHHL rabbits analyzed by high performance liquid chromatography. The Chol-fed NZW showed elevated β-VLDLs, and the WHHL rabbits showed increased LDLs and reduced HDLs. (b) Cumulative distribution of depth of coverage for whole-genome sequencing. The average depth of coverage was 13× for each individual rabbit. (c) Phylogenic tree of the rabbits. The tree was constructed on the basis of representative SNPs with the maximum likelihood method. Bootstrap values are marked on the branch. (d) Distribution of nucleotide diversity π. The statistics were calculated for every 100 kb sliding-window across the genome.
underwent a severe population bottleneck during artificial selection. The level of linkage disequilibrium was lowest in NZW rabbits and highest in WHHL rabbits (Supplementary Fig. S7), which is also in agreement with their breeding history.
Deleterious mutations in WHHL rabbits. Although WHHL rabbits are well-known for their LDLR
mutation as a cause of hypercholesterolemia5, it is possible that other deleterious mutations could rise to high frequency by genetic drift due to the extremely small population size of the breed. To search for such deleterious mutations possibly involved in cardiovascular diseases, we compiled a comprehensive gene list associated with cardiovascular diseases from both the knowledge database and human genome-wide association studies (Supplementary Fig. S8). Based on the functional annotations of SNPs and indels, we predicted deleterious mutations in the prior genes using the following criteria: 1) the mutation should alter the protein sequence; 2) for non-synonymous SNPs, it should have a SIFT score 0.4 WHHL rabbits harbor a larger proportion of deleterious mutations with high frequency than NZW rabbits, which supported our hypothesis that deleterious mutations were easier to accumulate in the WHHL breed. As the genetic diversity and hence the population size of JW rabbits were quite close to WHHL rabbits (Table 1), this result could only be observed when ΔAF > 0.8 between the two breeds. We focused on the putative deleterious mutations specifically enriched in WHHL rabbits with a criterion of ΔAF > 0.7 compared with both normal breeds, which resulted in 24 deleterious mutations in the prior genes in addition to the known 12-bp in-frame deletion in LDLR5 (Fig. 2b and Supplementary Table S1). One of the deleterious mutations was located in ALDH2, the activation of which was found to decrease aortic atherosclerosis14. It is well-known that a loss-of-function mutation in human ALDH2, E487K, causes alcohol flushing and is associated with an increased risk of cardiovascular diseases15. Similar to this mutation in humans, the deleterious mutation that we predicted in the WHHL rabbits, R99C, occurred at a conserved site, which was invariant among all the vertebrates we examined (Fig. 2c and Supplementary Fig. S9). The mutant allele frequency remained low in the normal rabbits (NZW 10%, JW 0%) but became fixed in WHHL rabbits (100%). Another putative deleterious mutation was located in VWF (Fig. 2b), the mediator of blood coagulation. A mutation at the same position in the human VWF protein was reported in patients with type I von Willebrand’s disease16. As a hypercoagulable state was found in WHHL rabbits17, the role of the deleterious mutation of VWF deserves further investigation. There Scientific Reports | 6:26942 | DOI: 10.1038/srep26942
3
www.nature.com/scientificreports/
Figure 2. Deleterious mutations in WHHL rabbits. (a) Proportion of deleterious mutation by difference in allele frequency (ΔAF) between WHHL and NZW or JW rabbits. (b) Putative deleterious mutations in WHHL rabbits with ΔAF > 0.7 compared with both normal rabbits. Colors show the density of SNPs from high (red) to low (blue). Genes harboring deleterious mutations are highlighted. (c) Non-synonymous mutations in ALDH2. The red cross indicates the locations of mutations. R99C is a putative deleterious mutation in WHHL rabbits. E487K is a known loss-of-function mutation in humans. Both mutations occur at highly conserved sites across vertebrates.
was also a deleterious mutation identified in OLR1 (Fig. 2b), and the gene was highly expressed in the atherosclerotic lesions of WHHL rabbits18. These results suggested that the deleterious mutations could function as genetic modifiers in the pathophysiology of WHHL rabbits.
Transcriptome profiling of WHHL and Chol-fed rabbits. We conducted deep transcriptome sequencing of different organs collected from both normal and hypercholesterolemic rabbits (Table 1 and Supplementary Figs S10 and 11). More than 77% of the assembled transcripts were isoforms of 15,760 known genes recorded in Ensembl (v76)19 (Supplementary Fig. S12). Samples originating from the same tissue were clustered together based on their gene expression profiles (Supplementary Fig. S13). We performed differential expression analysis for Chol-fed versus normal chow-fed NZW rabbits and WHHL versus JW rabbits (Supplementary Figs S14 and 15). The results showed that differentially expressed genes (DEGs) predominantly occurred in the aortas of both hypercholesterolemic models (2,719 and 1,627, respectively, false discovery rate [FDR]
